DESCRIPTION: This is a proposal for renewed funding of a long-term successful project to study the expression of histone genes and function of histones in Saccharomyces cerevisiae. The focus of the project has been on histones H3 and H4. The role of these proteins in three phenomena are to be studied. First, maintenance of genome integrity; a mutation replacing the four lysines in the N-terminal domain of H4 with glutamines (hhf1-10) increases spontaneous mutagenesis, apparently by increasing the occurrence of intrinsic DNA damage (as opposed to induced damage). Second, protein-protein interactions within the nucleosome; eliminating specific tyrosines in H4 interferes with interactions with the two H2B molecules in the nucleosome. Third, segregation of chromosomes; a mutation of H4, hhf1-20, exhibits defects in chromosome segregation. The lysines in the N-terminal domain may be replaced by lysines inserted seemingly at random locations, but not by arginines, suggesting that it is the regulation of charge (by acetylation and deacetylation of lysine) that regulates mutability, possibly by the charge density within the N- terminus of H4 regulating chromatin compaction. Dr. Smith will test if the arginines in the N-terminus contribute to this charge density, testing if eliminating the arginines exacerbates the phenotype of mutants retaining one lysine. He will also look for intragenic suppressors of the defect in hhf1-10 using doped oligonucleotide mutagenesis. He will also look for interactions between histones, hypothesizing that they may cooperate in modulating chromatin structure. A histone H3 mutation lacking all lysines in the N-terminus has a phenotype similar to hhf1-10. He will test cooperation in double mutants carrying both the H3 and H4 lysine-less mutations, looking for a more severe mutagenesis phenotype. If this supports cooperation, he will test mutations in H2A and H2B as well. To directly test the effect of these mutations on chromosome condensation he will use fluorescence in situ hybridization (FISH) to measure the physical distance between defined positions on various chromosomes in cycling cells, and in cells arrested at various steps of the cell cycle. Since hhf1-10 activates the RAD9 DNA-damage checkpoint pathway, which also blocks transit of START and G1/S, Dr. Smith will test if hhf1-10 exhibit a transient G1/S block as well. Finally, he will test if N-terminal domain mutants have defects in chromosome segregation, mitotic recombination, and specific pathways of DNA repair. For Aim 2, Dr. Smith will capitalize on his collaboration with Dr. E. N. Moudrianakis (who solved an X-ray structure of the nucleosome) to address sequence requirements of histone*histone interactions. One interaction mutant, the Y72G mutant of H4 which interferes with H4*H2B interaction, causes cell cycle arrest at START (all other histone mutations have a G2/M arrest). Dr. Smith has shown that this arrest results from lack of expression of the G1 cyclins, and hypothesizes that low expression results from poor expression of the CLN activators, Swi4 and Swi6, or directly from the chromatin structure of the CLN promoters. He will directly assess expression of the activators, and test the phenotype of double mutants between the Y72G mutant and swi4 or swi6. Second, he will test the existence of half-nucleosomes, predicted to be created during transcription through nucleosomal DNA. Aim 3 concerns chromosome segregation of histone H4 mutants and mutants in the CSE4 gene, identified by Dr. Smith as a dosage suppressor of the hhf1-20 chromosome segregation defect. Sequence comparisons suggested that Cse4p might be involved in kinetochore function. Dr. Smith will test the idea that Cse4p is required for kinetochore formation or function by testing if it is localized to centromeric regions, if histone H4 and Cse4p physically interact to form specialized centromeric nucleosomes, if this complex interacts with kinetochore proteins, and if formation of the Cse4p nucleosomes is regulated with respect to kinetochore assembly. Localization will be attempted by immunomicroscopy using Myc-epitope tagged Cse4p. Dual Myc and 6-His tags will be used to purify DNA associated with Cse4p; using a PCR assay, or using DNA-blotting to the Olson-Riles l clones, Dr. Smith will attempt to determine if centromeric DNA is overrepresented in this preparation. He will test an interaction between H4 and Cse4p first by attempting to identify reciprocal allele-specific suppressors, and second by attempt to reconstitute such a complex from purified H4 and Cse4p. Unusual sequences in Cse4p may be required for its kinetochore-specific function. Dr. Smith will test the role of these sequences by mutagenesis, or by the DNA shuffling technique of Stemmer (in which short segments of related genes are shuffled using a PCR approach). Dr. Smith will also test interactions between Cse4p and/or H4 and known segregation-related genes, and by using the two-hybrid approach to identify interacting gene products. Finally, he will test the hypothesis that H4 and Cse4p are necessary for morphogenesis of active kinetochores. He will seek dominant extragenic suppressors of hhf1-20 arrest. This is a continuation of an Aim from the previous funding period, during which 374 such mutants were obtained. During the next period, Dr. Smith will characterize the identified genes, confirming Mendelian segregation and suppression of H4 or Cse4p defects, and establishing complementation groups. Priority will be given to mutations which have independent chromosome segregation defects.